Anti cd84 antibodies, compositions comprising same and uses thereof

ABSTRACT

An isolated antibody comprising an antigen recognition domain which specifically binds CD84 and
         (i) down regulates the anti-apoptotic activity of stromal cells on chronic lymphocytic leukemia (CLL) cells; and/or   (ii) induces mobilization of CLL cells from the bone marrow.       

     Also provided are antibodies comprising antigen recognition domains comprising complementarity determining regions as indicated and uses thereof.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to anti CD84antibodies, compositions comprising same and uses thereof.

In normal individuals, the pool of peripheral lymphocytes is constant insize. The control of lymphoid homeostasis is the result of a very finebalance between lymphocyte production, survival, and proliferation.Survival factors have been shown to play a critical role in maintaininglymphocyte homeostasis.

Chronic lymphocytic leukemia, the most common leukemia in the Westernworld, is characterized by the progressive accumulation of CD5⁺ smallmature lymphocytes, in the peripheral blood, lymphoid organs and bonemarrow. The hallmark of the disease is decreased apoptosis, resulting inaccumulation of these malignant cells. Despite major progress in thelast few years in the understanding of the biology and pathophysiologyof the disease, as well as the development of better treatmentmodalities, CLL remains incurable in most patients, and even control ofthe disease requires aggressive treatment with significant side effects.A better understanding of the cellular events involved in thepathogenesis and progression of the disease should lead to more targetedand less toxic therapies, with early treatment in patients at risk,possibly enabling cure.

CD84 is a member of the CD2 subset of the immunoglobulin superfamily ofcell surface molecules. It is a single chain cell-surface protein withan extracellular portion of 199 aa, which contains four potentialN-glycosylation sites. The transmembrane region consists of 25 aa, andthe 83 aa cytoplasmic tail contains four tyrosines [delaFuente et al.Blood. 1997; 90:2398-2405]. The human CD84 is 57.3% identical to murineCD84. CD84 is predominantly expressed by B cells, T cells, platelets,monocytes, dendritic cells (DCs), and CD84 is also expressed early inhematopoiesis [Calpe et al. Advances in Immunology, Vol 97. 2008;97:177-250].

The present inventors have previously shown that the expression of CD84is significantly elevated from the early stages of the disease, and isregulated by macrophage migration inhibitory factor and its receptor,CD74. Activation of cell surface CD84 initiates a signaling cascade thatenhances CLL cell survival. Both downmodulation of CD84 expression andits immune-mediated blockade induce cell death in vitro and in vivo. Inaddition, analysis of samples derived from an on-going clinical trial,in which human subjects were treated with humanized anti-CD74(milatuzumab), shows a decrease in CD84 messenger RNA and protein levelsin milatuzumab-treated cells. This downregulation was correlated withreduction of Bcl-2 and Mcl-1 expression. Thus, overexpression of CD84 inCLL is an important survival mechanism that appears to be an early eventin the pathogenesis of the disease (Binsky-Ehrenreich et al. E. Pub.Feb. 25, 2013 Oncogene).

WO2010/035259 teaches CD84 as a regulator protein that is essential forthe survival of CLL cells. Based on this finding, the inventors ofWO2010/035259 have suggested the use of CD84 as a target for B-CLLtreatment and as a marker for the disease.

ADDITIONAL RELATED ART

U.S. Patent Application No. 20050027114 discloses methods of treatingdiseases such as chronic leukemia by agonizing or antagonizing anactivity of a CD84-like polypeptide.

U.S. Patent Application No. 20050025789 discloses the treatment orprophylaxis of tumors in patients, using a co-stimulatory polypeptide(e.g., CD84)-expressing tumor cell for producing a vaccine forincreasing the lytic activity of NK cells.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided an isolated antibody comprising an antigen recognitiondomain which specifically binds CD84 and:

-   (i) down regulates the anti-apoptotic activity of stromal cells on    chronic lymphocytic leukemia (CLL) cells; and/or-   (ii) induces mobilization of CLL cells from the bone marrow.

According to an aspect of some embodiments of the present inventionthere is provided an isolated antibody comprising an antigen recognitiondomain comprising complementarity determining regions as set forth inSEQ ID NOs: 1, 2, 3, 4, 5 and 6 (B4), wherein the antibody specificallybinds CD84.

According to an aspect of some embodiments of the present inventionthere is provided an isolated antibody comprising an antigen recognitiondomain comprising complementarity determining regions as set forth inSEQ ID NOs: 7, 8, 9, 10, 11 and 12 (B1), wherein the antibodyspecifically binds CD84.

According to some embodiments of the invention, the isolated antibodydown regulates the anti-apoptotic activity of stromal cells on chroniclymphocytic leukemia (CLL) cells.

According to some embodiments of the invention, the isolated antibodyinhibits the secretion of CCL3 from CLL cells.

According to some embodiments of the invention, the isolated antibodyinhibits the expression of BCL-2 in CLL and stromal cells.

According to some embodiments of the invention, the isolated antibodyinhibits the expression of stroma Bcl-2, thereby reducing theanti-apoptotic effect of the stroma on CLL.

According to some embodiments of the invention, the isolated antibodyreduces the expression of cytokine like IL-6 and IL-8 in stromal cellsthat supports CLL survival.

According to some embodiments of the invention, the isolated antibodyinduces mobilization of CLL cells from the bone marrow.

According to some embodiments of the invention, the isolated antibody isan IgG.

According to some embodiments of the invention, the antibody is ahumanized antibody or a chimeric antibody.

According to some embodiments of the invention, the antibody is abispecific antibody.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising the isolatedantibody and a pharmaceutically acceptable carrier or diluent.

According to an aspect of some embodiments of the present inventionthere is provided a method of inducing apoptosis in B cells of a subjecthaving a B cell malignancy, the method comprising administering to thesubject a therapeutically effective amount of the antibody, therebyinducing apoptosis in B cells of the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a B cell malignancy in a subjectin need thereof, the method comprising administering to the subject atherapeutically effective amount of the antibody, thereby treating the Bcell malignancy.

According to an aspect of some embodiments of the present inventionthere is provided use of the antibody in the manufacture of a medicamentidentified for the treatment of a B cell malignancy.

According to some embodiments of the invention, the B cell malignancy isselected from the group consisting of a lymphoma, a leukemia and amyeloma.

According to some embodiments of the invention, the B cell malignancy isselected from the group consisting of a Hodgkin's Lymphoma, anon-Hodgkin's Lymphoma, a Diffuse large B-cell lymphoma, a B-cellchronic lymphocytic leukemia (B-CLL)/chronic lymphoid leukemia (CLL), aChronic lymphocytic leukemia/small lymphocytic lymphoma, a chronicmyelocytic leukemia (CML), an Extranodal marginal zone B-celllymphoma—mucosa-associated lymphoid tissue lymphoma, a Follicularlymphoma, a Mantle cell lymphoma, a Nodal marginal zone B-cell lymphoma,a Burkitt's lymphoma, a Hairy cell leukemia, a Primary central nervoussystem lymphoma, a Splenic marginal zone B-cell lymphoma, aLymphoplasmocytic lymphoma, a Primary mediastinal B-cell lymphoma, amultiple myeloma, an acute lymphocytic leukemia (ALL), an acutelymphoblastic pre-B cell leukemia, a plasma cell leukemia, a pre-B cellleukemia, an early pre-B cell leukemia and a pre-B acute lymphoblastoidleukemia.

According to some embodiments of the invention, the B cell malignancy isa B-CLL.

According to an aspect of some embodiments of the present inventionthere is provided a method of diagnosing B-CLL in a subject in needthereof, the method comprising:

(a) contacting a biological sample of the subject with the antibody ofany one of claims 1-12 under conditions which allow the formation ofimmunocomplexes between CD84 isoform C (SEQ ID NO: 14) and the antibody;and

(b) determining a level of the immunocomplexes in the biological sample,wherein an increase in level of the immunocomplexes beyond apredetermined threshold with respect to a level of the immunocomplexesin a biological sample from a healthy individual is indicative of theB-CLL.

According to some embodiments of the invention, the determining iseffected at the protein level.

According to some embodiments of the invention, the method furthercomprises corroborating the diagnosis using a diagnostic assay selectedfrom surface marker expression distinctive of the CD84 isoform c,karyotype analysis and germline mutations.

According to some embodiments of the invention, an isolatedpolynucleotide comprising a nucleic acid sequence encoding the antibody.

According to some embodiments of the invention, the isolatedpolynucleotide is as set forth in SEQ ID NO: 26 or 27.

According to an aspect of some embodiments of the present inventionthere is provided a method of identifying a putative drug against a Bcell malignancy, the method comprising:

(a) treating a model animal having a B cell malignancy with a testagent; and

(b) detecting B cell malignancy cells in a peripheral tissue versus in abone marrow of the model animal treated as in (a), wherein an increasein the ratio as compared to the ratio prior to the treatment isindicative that the test agent is a putative drug against the B cellmalignancy.

According to some embodiments of the invention, the test agent is ananti CD84 antibody.

According to some embodiments of the invention, the model animal isselected from the group consisting of a xenograft model induced bytransplanting human CLL in mice and the Eu-TCL1 murine model.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-D are scatter plots showing the ability of the blockinganti-CD84 antibody of hybridoma B4 to induce CLL cell death. FIGS.1A-B—1×10⁷ CLL cells were incubated with anti-CD84 from the B4 hybridoma(5 μg/ml), or a control antibody (IgG2a). 24 h later, CLL cell death wasanalyzed by Annexin 7AAD staining. Presented are representative plotsout of 10 independent experiments. FIGS. 1C-D—1.6×10⁶ CLL cells wereco-cultured with 1×10⁵ M210B4, in the presence or absence of theanti-CD84 from the B4 hybridoma (5 μg/ml), or a control antibody.Following 48 h, CLL cell death was determined with Annexin 7AADstaining. Presented are representative plots out of 3 independentexperiments.

FIGS. 2A-C are bar graphs showing the ability of the anti-CD84 from theB4 hybridoma to mediate an anti-apoptotic effect on M210B4 bone marrowstromal cells in co cultures. In addition, homophilic interactions ofCD84 expressed on CLL and stroma cells cause cytokine secretion from thestroma in order to further support the survival of CLL cells. M210B4cells (1×10⁵) were co cultured with CLL cells in the presence or absenceof the anti-CD84 from the B4 hybridoma or an isotype control antibodies.48 h later, cells were harvested and Bcl-2, IL-6 and VCAM-1 mRNA levelswere analyzed by qRT-PCR. Results presented are a summary of nindependent experiments and are expressed as a fold of change inexpression in co-cultured M210B4 cells compared to co-cultured cellsblocked for CD84 and to M210B4 cells (stroma only), which was definedas 1. (FIG. 2A) Fold of change in expression of Bcl-2, n=4, P=0.037(FIG. 2B) fold of change in expression of IL6, n=5, P=0.037 (FIG. 2C)fold of change in expression of VCAM-1 n=6, P=0.25.

FIG. 3 is a bar graph showing that CD84 regulates CCL3 secretion in cocultures. 1.6×10⁶ CLL cells were cultured with 1×10⁵ M210B4 cells in thepresence or absence of the anti-CD84 from the B4 hybridoma or an isotypecontrol antibody (5 μg/ml). 48 h later, CCL3 level in the conditionedmedium was analyzed by ELISA. Graphs show fold of change in CCL3 levelsfollowing CD84 blockage. The graph summarizes 6 experiments, *P=0.02.

FIGS. 4A-H show that the antibodies of some embodiments of the inventionare superior in killing B-CLL cells as compared to the F8 antibody, asdetermined by Magic Red apoptosis assay. FIGS. 4A-D—Purified CLL cellswere cultured in 24-well plates at a density of 1×10⁷ cells/well in RPMImedium supplemented with 10% FCS, 2 mM glutamate, 100 U/ml penicillin,100 μg/ml streptomycin, with or without supernatant derived fromanti-CD84 hybridoma (F8/B1/B4) or control hybridoma for 24 h. Cells werecentrifuged, washed and incubated with Magic Red (ImmunochemistryTechnology) according to the manufacturer's instructions, at 37° C. for1 h. Then, Magic Red staining was measured by FACS analysis. FIGS.4E-H—Ramos cells were cultured in 24-well plates at 1×10⁷ cells/well inRPMI medium supplemented with 10% FCS, 2 mM glutamate, 100 U/mlpenicillin, 100 μg/ml streptomycin, with or without supernatant derivedfrom anti-CD84 hybridoma (F8/B1/B4) or control hybridoma for 24 h. Cellswere centrifuged, washed and incubated with Magic Red (ImmunochemistryTechnology) according to the manufacturer's instructions, at 37° C. for1 h. Then, Magic Red staining was measured by FACS analysis.

FIGS. 5A-B are scatter plots showing reduced number of CLL cells in BMof CD84KO mice. 1×10⁷ CLL cells were stained with CFSE and injected i.v.into C57BL/6 WT or CD84KO mice. After 1 h (FIG. 5B) and 4 h (FIG. 5A),mice were sacrificed and number of CLL cells in spleens and BM wereanalyzed by FACS. The graphs show the ratio of labeled cells recoveredfrom the BM to number of cells recovered from the spleens of (FIG. 5A)16 mice in each group. *P=0.0005 or (FIG. 5B) 6 mice in each group, nostatistical significant difference between groups.

FIGS. 6A-M show that B1 and B4 antibodies recognize murine cells derivedfrom TCL-1 mice. FIG. 6A is a histogram showing CD84 expression (blackline) or staining with an isotype matched control antibody (grey line)on B220+CD5+cells derived from TCL-1 mice; FIGS. 6B-D are dot plotsshowing the percent of the B220+CD5+ in splenic cells derived from 18,11 and 6 months old TCL-1 mice; and FIGS. 6E-M are dot plots. 1×10⁷B220+CD5+ cells derived from splenic TCL-1 mice were incubated with theB1 or the B4 or an isotype matched control (IgG2a) antibodies. 48 hlater, cell death was analyzed by Annexin-7AAD staining.

FIGS. 7A-D show evaluation of the therapeutic potential of the B1 and B4antibodies in different tumor cell lines. FIG. 7A illustrate mRNA levelsof CD84 and actin which were analyzed in different solid tumors: MDA435(Human breast cancer), PC-3 (Human prostate cancer), Hela (Humancervical cancer), A375 (Human malignant melanoma), A549 (Human lungadenocarcinoma), PAC1 (Human Pancreas cancer) and N87 (Human gastriccancer); FIG. 7B is a histogram showing CD84 expression (black line) orstaining with an isotype matched antibody (grey line) on Daudi cells;FIGS. 7C-D show 1×10⁷ Daudi cells which were incubated with the B1 orthe B4 antibody, or a control antibody (IgG2a). 48 h later, cell deathwas analyzed by Annexin-7AAD staining. The graph summarizes 7independent experiments, showing fold of change in Annexin positivecells compared to Daudi cells incubated with an isotype matched controlantibody.

FIGS. 8A-F show an elevated expression of CD84 in CLL cells. (FIG. 8A) Bcells derived from healthy subjects (normal; N=4), as well as early-(N=6) and advanced- (N=6) stage CLL patients were purified and examinedfor CD84 expression. CD84 and actin mRNA were analyzed by RT-PCR; (FIG.8B) qRT-PCR was performed using primers for CD84 and RP-2. Results areexpressed as fold-change in CD84 mRNA in CLL cells compared to normal Bcells, which was defined as 1. The graph summarizes results of threenormal donors, and seven CLL patients; (FIGS. 8C-E) Histograms showingCD84 expression (grey line) or staining with secondary Ab alone (dottedline) in normal B cells and CLL cells. The graphs summarize the resultsof 4 normal donors and 18 CLL patients in percent or MFI; (FIG. 8F) Dotplots show CD84 and CD5 expression on CD19 positive cells normal (N=3)and CLL (N=4) cells.

FIGS. 9A-J show that B1 and B4 antibodies do not induce apoptosis innormal B cells. 1×10⁷ B220+ splenic cells derived from 6, 8 and 11months old C57BL/6 mice were incubated with the B1 or the B4 or acontrol (IgG2a) antibodies. 48 h later, cell death was analyzed byAnnexin-7AAD staining. Graphs summarize 3 independent experiments,showing fold of change in the live B220+ cells incubated with B 1, B4 oran isotype matched control antibody.

FIGS. 10A-B show that CD84 is expressed on stroma cells. (FIG. 10A) CLL,NLC and M210B4 cells were purified and examined for CD84 expression.CD84 and actin mRNA were analyzed by RT-PCR; (FIG. 10B) Histograms showCD84 expression (black line) or staining with secondary Ab alone (greyline) in CLL, NLC and M210B4 cells.

FIGS. 11A-G show that CD84 expressed on stroma cells regulate survivalof CLL cells. (FIGS. 11A-C) 1.6×10⁶ CLL cells were co-cultured with1×10⁵ M210B4, in the presence or absence of the anti-CD84 B4 hybridoma(5 μg/ml), or a control antibody. After 24 h CLL cell death wasdetermined with Annexin 7AAD staining. Presented are representativeplots out of 3 independent experiments; (FIGS. 11D-G) siRNA(ON-TARGETplus SMARTpool, Human CD84 (NM_003874), Dharmacon) for CD84 ora control scrambled siRNA was transfected into 0.625×10⁵ M210B4 cells.18 h later 1×10⁶ CLL cells were added to the culture. 48 h later, CLLcells were collected. Representative dot plots are demonstrated. Graphsummarizes 9 independent experiments showing relative live CLL cells(average+standard error). *P=0.04, **P=3.1*10−10.

FIGS. 12A-D show that blocking CD84 on stroma with the B1 and B4antibodies reduces survival of CLL cells. 0.625×10⁵ M210B4 cells wereincubated with the B1 or the B4 or a control antibody (IgG2a)antibodies. After 1 h, the antibodies were washed and 1×10⁶ CLL cellswere added. 48 h later cell survival was measured by Annexin-7AADstaining. The graph summarizes 3 independent experiments, showing foldof change in live CLL cells compared to CLL cells incubated with anisotype control antibody treated stroma.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to anti CD84antibodies, compositions comprising same and uses thereof.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulationof CD5+ B lymphocytes in peripheral blood, lymphoid organs and bonemarrow. The main feature of the disease is accumulation of the malignantcells due to decreased apoptosis. CD84 belongs to the signalinglymphocyte activating molecule family of immunoreceptors, and has anunknown function in CLL cells. The present inventors have previouslyshown that the expression of CD84 is significantly elevated from theearly stages of the disease, and is regulated by macrophage migrationinhibitory factor and its receptor, CD74. Activation of cell surfaceCD84 initiates a signaling cascade that enhances CLL cell survival. Bothdownmodulation of CD84 expression and its immune-mediated blockadeinduce cell death in vitro and in vivo. In addition, analysis of samplesderived from an on-going clinical trial, in which human subjects weretreated with humanized anti-CD74 (milatuzumab), shows a decrease in CD84messenger RNA and protein levels in milatuzumab-treated cells. Thisdownregulation was correlated with reduction of Bcl-2 and Mcl-1expression. Thus, overexpression of CD84 in CLL is an important survivalmechanism that appears to be an early event in the pathogenesis of thedisease (Binsky-Ehrenreich et al. E. Pub. Feb. 25, 2013 Oncogene,WO2010/035259). These findings suggest novel therapeutic strategiesbased on the blockade of this CD84-dependent survival pathway.

The present inventors have now realized that homophilic interactionsbetween CD84 expressed in B-CLL cells and stromal cells induce asurvival signal in the cancer cells and independently retain the cellsin the stromal environment of the bone marrow (see FIGS. 5A-B). Thepresent inventors have further uncovered that CD84 is expressed onvarious types of lymphoma cells (FIG. 7B and FIG. 10A) as well as ondifferent types of stromal cells. This suggests that CD84 might beinvolved in cell-cell interaction between CLL cells and theirmicroenvironment (see FIG. 10A).

Thus, while screening for novel antibodies that block CD84-dependentsurvival pathway of CLL and induce mobilization of the CLL cells fromthe bone marrow, the present inventors have identified novel antibodiesthat induce apoptosis of CLL cells and protect against theanti-apoptotic activity and adhesive activity of stromal cellsco-cultured therewith. Antibodies derived from the B1 and B4 hybridomaswere able to downregulate secretion of CCL3 from CLL (Example 4) andinduce apoptosis of purified CLL cells and Ramos cell line in a superiormanner as compared to a previously isolated anti CD84 antibody describedin WO2010/035259 (Example 5). These results place the present antibodiesas important immunotherapy against CLL.

Thus, according to an aspect of the invention there is provided a methodof identifying a putative drug against a B cell malignancy (e.g. B-CLL),the method comprising:

(a) treating a model animal having B cell malignancy (e.g. B-CLL) with atest agent; and

(b) detecting B cell malignancy (e.g. B-CLL) cells in a peripheraltissue versus in a bone marrow of the model animal treated as in (a),wherein an increase in the ratio as compared to the ratio prior to thetreatment is indicative that the test agent is a putative drug against Bcell malignancy (e.g. B-CLL).

As used herein “a model animal having a B cell malignancy” refers to amammal such as a non-human mammal which exhibits clinical manifestationsof a B cell malignancy including B-CLL. The model may be a spontaneousmodel, a transgenic model or a xenograft model in which human B cellmalignant cells (e.g. B-CLL cells) are transplanted.

Kurtova et al. Blood. 2009 Nov. 12; 114(20):4441-50 (herein incorporatedby reference) describes several transgenic mouse models for B-CLL.Investigations of these mouse models revealed that deregulation of threepathways, Tcl1-Akt pathway, TNF-NF-kB pathway, and Bcl2-mediatedanti-apoptotic pathway, result in the development of B-CLL. Whilederegulation of TCL1 alone caused a B-CLL phenotype in mice,overexpression of Bcl2 required aberrantly activated TNF-NF-kB pathwaysignaling to yield the disease phenotype.

According to a specific embodiment, the model animal is selected fromthe group consisting of a xenograft model induced by transplanting humanCLL in mice and the Eu-TCL1 murine model.

As used herein “a test agent” refers to a molecule which decreases CD84expression or activity. The test agent can be a small molecule, anucleic acid silencing agent (e.g., siRNA or antisense), an antibody ora peptide (e.g., a soluble CD84 molecule which interferes with CD84homophilic interactions, such as described in WO2010/035259).

According to a specific embodiment the test agent is an anti CD84antibody.

Detection of malignant B cells (e.g. B-CLL cells) can be done usingmethods which are well known in the art and mainly depend on the modelused.

Thus, when using the xenograft model, monitoring mobilization can bedone by detecting human moieties in the mouse organs. Examples include,but are not limited to analyzing cell surface markers (human CD19) orpre-stained (CFSE+) cells.

Alternatively or additionally the human malignant B cells (e.g. B-CLLcells) can be stained (e.g., fluorescently, radio-isotope staining)prior to transplantation for ease of detection.

Examples of peripheral tissues in which the amount of malignant B cells(e.g. B-CLL cells) can be monitored include, but are not limited to,spleen, peritoneal cavity, peripheral blood, lymph nodes and bone marrow(Durig J et al, Cancer Res 2007; 67: 8653-8661).

The ratio of malignant B cells (e.g. B-CLL cells) in a peripheral tissueversus in a bone marrow is compared to a control animal. A controlanimal may be the same animal prior to treatment or the same animaltreated with a control agent (e.g., PBS or saline) which doesn't affectmalignant B cell (e.g. B-CLL) mobilization.

As used herein an “increase” refers to a statistically significantincrease. Thus the increase can be by at least 2, 5%, 10%, 20%, 50% ormore.

Using the present methodology anti CD84 antibodies can be isolated.

Thus, according to an aspect of the invention there is provided anisolated antibody comprising an antigen recognition domain whichspecifically binds CD84 and,

-   (i) down regulates the anti-apoptotic activity of stromal cells on    chronic lymphocytic leukemia (CLL) cells; and/or (i.e., additionally    or alternatively)-   (ii) induces mobilization of CLL cells from the bone marrow (to    peripheral organ/tissues).

As used herein “down regulates” refers to a statistically significantreduction. Thus the decrease can be by at least 2, 5%, 10%, 20%, 50% ormore.

As used herein “stromal cells” refers to adherent cells which reside inthe bone marrow, also referred to as “marrow stromal cells”. Examples ofmarrow stromal cell lines and primary stromal cell lines are providedherein below and in Kurtova et al. supra.

The reduction in the anti-apoptotic activity of stromal cells can bedetected by reduction in survival factors such as IL-6 (in stroma), IL-8(in stroma) and Bcl-2 (B-CL-2, which is reduced in both the stroma andCLL).

Thus, according to an aspect of the invention there is provided anisolated antibody comprising an antigen recognition domain comprisingcomplementarity determining regions (CDR as set forth in SEQ ID NOs: 1,2, 3, 4, 5 and 6 (B4), wherein the antibody specifically binds CD84.

According to a specific embodiment the isolated antibody comprises SEQID NOs: 4 (CDR1), 5 (CDR2) and 6 (CDR3), (sequentially arranged from Nto C on a light chain of the protein) and 1 (CDR1), 2 (CDR2) and 3(CDR3) (sequentially arranged from N to C on a heavy chain of theprotein) (Clone B4).

Alternatively, there is provided an isolated antibody comprising anantigen recognition domain comprising complementarity determiningregions as set forth in SEQ ID NOs: 7, 8, 9, 10, 11 and 12 (B1), whereinthe antibody specifically binds CD84.

According to a specific embodiment the isolated antibody comprises SEQID NOs: 10 (CDR1), 11 (CDR2) and 12 (CDR3), (sequentially arranged fromN to C on a light chain of the protein) and 7 (CDR1), 8 (CDR2) and 9(CDR3) (sequentially arranged from N to C on a heavy chain of theprotein) (Clone B1).

The antibodies of the present invention having the above-mentioned CDRs,are collectively referred to as “the anti-CD84 antibodies of the presentinvention”.

As used herein the term “CD84” refers to an expressed isoform of theCD84 gene. Examples include but are not limited to Q9UIB8-1, Q9UIB8-2,Q9UIB8-3, Q9UIB8-4, Q9UIB8-5, Q9UIB8-6 and Q9UIB8-7.

According to a specific embodiment, the CD84 refers to CD84 isoform C,an isoform of CD84 which is assigned with Accession Numbers AF054815.1NP_003865.1 (NM_003874, Q9UIB8-3) or SEQ ID NOs: 13 or 14.

The general affinity of the anti CD84 antibody is preferably higher thanabout, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M and as such is stableunder physiological (e.g., in vivo) conditions.

According to a specific embodiment the affinity is preferably higherthan (i.e., at least) about, 10⁻⁸ M or 10⁻⁹ M, e.g., 1-50×10⁻⁹ M,1-100×10⁻⁹ M, 0.5-50×10⁻⁹ M or 0.5-100×10⁻⁹ M.

As used herein the term “isolated” refers to a level of purity such thatthe protein of the invention is the predominant form (e.g., more than50%) in the preparation. In other words, other antibodies which arecharacterized by low or no affinity to CD84 (exceeding the above values)are altogether present in the preparation in less than 50% of the totalantibody molecules of the preparation. According to a specificembodiment, the anti CD84 is isolated from the physiological embodimente.g., from the body (e.g., human or animal). According to a specificembodiment, the term isolated also means isolated from a library, suchas a phage display library or hybridoma lines or libraries.

The antibody of the present invention typically binds to theextracellular portion of CD84 (SEQ ID NO: 15).

As used herein, the term “epitope” refers to any antigenic determinanton an antigen to which the paratope of an antibody binds.

Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or carbohydrate side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics.

The term “antibody” as used in this invention includes intact moleculesas well as functional fragments thereof, such as Fab, F(ab′)2, and Fvthat are capable of binding to macrophages. These functional antibodyfragments are defined as follows: (1) Fab, the fragment which contains amonovalent antigen-binding fragment of an antibody molecule, can beproduced by digestion of whole antibody with the enzyme papain to yieldan intact light chain and a portion of one heavy chain; (2) Fab′, thefragment of an antibody molecule that can be obtained by treating wholeantibody with pepsin, followed by reduction, to yield an intact lightchain and a portion of the heavy chain; two Fab′ fragments are obtainedper antibody molecule; (3) (Fab′)2, the fragment of the antibody thatcan be obtained by treating whole antibody with the enzyme pepsinwithout subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragmentsheld together by two disulfide bonds; (4) Fv, defined as a geneticallyengineered fragment containing the variable region of the light chainand the variable region of the heavy chain expressed as two chains; and(5) Single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain and the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of producing polyclonal and monoclonal antibodies as well asfragments thereof are well known in the art (See for example, Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, 1988, incorporated herein by reference).

Antibody fragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by conventional methods. For example, antibody fragments canbe produced by enzymatic cleavage of antibodies with pepsin to provide a5S fragment denoted F(ab′)2. This fragment can be further cleaved usinga thiol reducing agent, and optionally a blocking group for thesulfhydryl groups resulting from cleavage of disulfide linkages, toproduce 3.5S Fab′ monovalent fragments. Alternatively, an enzymaticcleavage using pepsin produces two monovalent Fab′ fragments and an Fcfragment directly. These methods are described, for example, byGoldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein, which patents are hereby incorporated by reference intheir entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)].Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Fv fragments comprise an association of VH and VL chains. Thisassociation may be noncovalent, as described in Inbar et al. [Proc.Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise VH and VL chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the VH and VLdomains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by [Whitlow andFilpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426(1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No.4,946,778, which is hereby incorporated by reference in its entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry[Methods, 2: 106-10 (1991)].

According to a specific embodiment, the antibody is a monoclonalantibody of any subtype e.g., IgG, IgM, IgA etc. According to a specificembodiment the antibody is IgG1 or IgG4.

According to a specific embodiment, the antibody is an IgG2a (e.g., B4,B1) or IgG1 (e.g., B1). Humanized forms of non-human (e.g., murine)antibodies are chimeric molecules of immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesform a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introduction of human immunoglobulinloci into transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10,: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar,Intern. Rev. Immunol. 13, 65-93 (1995).

It will be appreciated that the CDR sequences described herein can beimplemented in a bispecific antibody configuration.

As used herein “bispecific” or “bifunctional” antibody, refers to anartificial hybrid antibody having two different heavy/light chain pairsand two different binding sites. Bispecific antibodies can be producedby a variety of methods including fusion of hybridomas. See e.g.,Songsivilai and Lachmann (1990) Clin. Exp. Immunol. 79:315-321; Kostelnyet al. (1992) J. Immunol. 148:1547-1553. The bispecific antibody maybind CD84 and another target which is expected to cooperate with CD84 inbiological processes, such as apoptosis, or may increase specificity tothe malignant B cell (e.g. B-CLL). Examples of such targets include, butare not limited to, CD74, CD19, CD5, SLAM family receptors (like NTB-A;SLAMF7). Alternatively or additionally, the bispecific antibody may bindCD84 at an epitope, which is distinctive of the epitope to which the B1or B4 antibodies bind. Alternatively or additionally, the bispecificantibody may include the B1 and B4 CDRs.

According to a specific embodiment the antibody inhibits CD84 homophilicinteractions.

As used herein “CD84 homophilic interactions” refer to the ability ofCD84 to strongly self-associate with a Kd in the submicromolar range;the association is driven by the Ig-V domain, forming an orthogonalhomophilic dimer. According to a specific embodiment the homophilicinteractions are between the CD84 on the CLL and the CD84 on the stromalcells as well as between CD84 on CLL cells.

Methods of determining CD84 homophilic interactions include, but are notlimited to siRNA of CD84 on the stroma, knockout mice of CD84. The CD84homophilic interactions were demonstrated previously in [Yan et al.,PNAS 2007].

It has been previously suggested that CLL cells are able to activelymanipulate their microenvironment [Binder M et al, PLoS One. 2012.5(12):e15992, Neil E Kay et al, Leuk Res. 2007 July; 31(7): 899-906 ].Specifically, it has been shown that CLL cells induce anti-apoptoticeffects on their stromal counterparts in culture. The CLL-stoma cellinteraction induces the expression and secretion of specific cytokines(i.e IL-6, IL-8) from the stroma and induces the expression of specificadhesion molecules like ICAM-1, while the expression of other adhesionmolecules remains unchanged (i.e VCAM-1) [Plander Metal, Annals ofHematology. 2011. 90(12):1381-90].

According to an alternative or an additional embodiment, the antibody iscapable of down regulating the anti-apoptotic activity of stromal cellson chronic lymphocytic leukemia (CLL) cells as well as that of the CLLcells on the stromal cell.

As shown in the Examples section which follows, CLL cells cultured withstromal cells in the presence or absence of a CD84 antibody of someembodiments of the invention showed a reduction of the anti-apoptoticgene Bcl-2 (FIG. 2A) and the cytokine IL-6 (FIG. 2B) repeats. Incubationwith CLL cells elevated Bcl-2 and IL-6 mRNA levels in stromal cells,while no change in VCAM-1 message were observed.

Interestingly, CD84 blockage reduced Bcl-2 (FIG. 2A) and IL-6 (FIG. 2B)message levels, while VCAM-1 levels were not affected by this blockage(FIG. 2C). Identification of changes in gene expression (mRNA level) orprotein synthesis/secretion can be done using methods which are wellknown in the art. mRNA expression can be quantified by RT-PCR or realtime quantitative PCR, secretion of cytokines can be detected usingELISA in culture medium.

As used herein the term “inhibiting” or “decreasing” refers to astatistically significant decrease in gene expression (e.g., Bcl-2) orprotein secretion (e.g., IL-6, IL-8 or CCL3) by at least 10%, 20%, 30%,40%, 50%, 60%, 80% or more.

Due to its ability to bind CD84 and inhibit its function (in at leastone the modes described above), the anti CD84 antibody of someembodiments of the invention is also referred to as a “blockingantibody” or a “neutralizing antibody” and as such can be used intherapeutic applications.

Thus, according to an aspect of the invention, there is provided amethod of inducing apoptosis in B cells of a subject having a B cellmalignancy (e.g. B-CLL), the method comprising administering to thesubject a therapeutically effective amount of the anti CD84 antibody asdescribed herein, thereby inducing apoptosis in B cells of the subject.

Accordingly, the present teachings also contemplate a method of inducingapoptosis in CLL cells. The method comprising contacting the CLL cellswith the antibody of the present invention (described above), therebyinducing apoptosis of the CLL cells.

According to an embodiment of this aspect of the invention, the methodis effected in vivo.

According to an embodiment of this aspect of the invention, the methodis effected ex vivo.

According to an embodiment of this aspect of the invention, the methodis effected in-vitro.

As used herein “inducing apoptosis” refers to increasing the level ofapoptosis in treated cells by at least 2%, 5%, 10%, 20%, 30%, 40%, 50%,70% or even more.

Methods of determining apoptosis include but are not limited to, Annexinstaining, and magic red staining. Specifics of such methods aredescribed in the Examples section which follows.

According to another aspect there is provided a method of treating B-CLLin a subject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of the anti CD84 antibody asdescribed herein, thereby treating B-CLL.

Yet according to another aspect there is provided use of the anti CD84antibody as described herein in the manufacture of a medicamentidentified for the treatment of a B cell malignancy.

As used herein, the term “subject” or “subject in need thereof” refersto a mammalian e.g., human subject, male or female at any age, who hasbeen diagnosed with a B cell malignancy.

As used herein the term “B cell malignancy” refers to a malignancy ofhematopoietic or lymphoid tissues involving B lymphocytes of any subtypeor stage of differentiation (e.g. early pre-B cells, pre-B cells, matureB cells, plasma cells).

According to one embodiment, the B cell malignancy comprises a lymphoma,a leukemia or a myeloma.

Such diseases include, but are not limited to, Hodgkin's Lymphoma,non-Hodgkin's Lymphoma, Diffuse large B-cell lymphoma (DLBCL), B-cellchronic lymphocytic leukemia (B-CLL)/chronic lymphoid leukemia (CLL),Chronic lymphocytic leukemia/small lymphocytic lymphoma, a chronicmyelocytic leukemia (CML), an Extranodal marginal zone B-celllymphoma—mucosa-associated lymphoid tissue lymphoma, a Follicularlymphoma, a Mantle cell lymphoma, a Nodal marginal zone B-cell lymphoma,a Burkitt's lymphoma, a Hairy cell leukemia, a Primary central nervoussystem lymphoma, a Splenic marginal zone B-cell lymphoma, aLymphoplasmocytic lymphoma, a Primary mediastinal B-cell lymphoma,multiple myeloma, Acute lymphocytic leukemia (also known as acutelymphoblastic leukemia or ALL), acute lymphoblastic pre-B cell leukemia,plasma cell leukemia, pre-B cell leukemia (e.g. pre-B ALL), early pre-Bcells ALL (e.g. early pre-B ALL) or pre-B acute lymphoblastoid leukemia.

According to one embodiment, the B cell malignancy is B-CLL.

As used herein the term “B-CLL” or “CLL” refers to an abnormalneoplastic proliferation of B-cells. CLL is considered to be identicalto a disease called small lymphocytic lymphoma (SLL), a type ofnon-Hodgkin's lymphoma which presents primarily in the lymph nodes. TheWorld Health Organization considers CLL and SLL to present differentstages of the same disease [Chiorazzi N, Rai K R, Ferrarini M (2005).“Chronic lymphocytic leukemia”. N. Engl. J. Med. 352 (8): 804-15].

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

The antibodies of the present invention can be administered to thesubject per se, or in a pharmaceutical composition where it is mixedwith suitable carriers or excipients.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the agent accountable forthe biological effect (i.e., down regulation in CD84 activity orexpression).

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular,intracardiac, e.g., into the right or left ventricular cavity, into thecommon coronary artery, intravenous, intraperitoneal, intranasal, orintraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

The term “tissue” refers to part of an organism consisting of anaggregate of cells having a similar structure and/or a common function.Examples include, but are not limited to, brain tissue, retina, skintissue, hepatic tissue, pancreatic tissue, bone, cartilage, connectivetissue, blood tissue, muscle tissue, cardiac tissue brain tissue,vascular tissue, renal tissue, pulmonary tissue, gonadal tissue,hematopoietic tissue.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients effective to prevent, alleviate or amelioratesymptoms of a disorder (e.g., a B cell malignancy including B-CLL) orprolong the survival of the subject being treated. In a specificembodiment, the therapeutically effective amount is sufficient to induceapoptosis of B cells of a B cell malignancy (e.g. induce apoptosis ofB-CLL cells).

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals.

A B-CLL animal model such as the NOD-SCID mouse chimera as describedpreviously [Shimoni A, Marcus H, Canaan A, et al. A model for humanB-chronic lymphocytic leukemia in human/mouse radiation chimera:evidence for tumor-mediated suppression of antibody production inlow-stage disease. Blood. 1997; 89:2210-2218], can be used to determinetherapeutic efficacy of the antibodies of the present invention in vivo.Human peripheral blood mononuclear cells from B-CLL patients atdifferent stages of the disease are transferred by intraperitonealinjection. This system supports long term survival of the human tumorcells. Chimeric mice are treated with the antibodies of the presentinvention for different periods of time, and the effect on grafting ofthe cells and survival is then assessed.

The data obtained from these in vitro and cell culture assays and animalstudies can be used in formulating a range of dosage for use in human.The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provide(tissue) levels of the active ingredient are sufficient to induce orsuppress the biological effect (minimal effective concentration, MEC).The MEC will vary for each preparation, but can be estimated from invitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as is further detailed above.

To improve therapeutic efficacy, antibodies of the present invention canbe further administered along with conventional therapy for a B cellmalignancy (e.g. for B-CLL) such as chemotherapy, radiotherapy,biological therapy e.g., immunotherapy or bone marrow transplantation.

Following is a non-limiting list of examples of conventional therapiesfor B-CLL.

Purine analogs—fludarabine or chlorambucil are generally used in thiscategory of treatments. Monoclonal antibodies—Monoclonal antibodies suchas alemtuzumab (directed against CD52) and rituximab (directed againstCD20) are generally used in this category of treatments.

Combination chemotherapy—Combination chemotherapy options are typicallyused in newly-diagnosed and relapsed CLL. Recently, randomized trialshave shown that combinations of purine analogues (fludarabine) withalkylating agents (cyclophosphamide) produce higher response rates and alonger progression-free survival than single agents: e.g., FC(fludarabine with cyclophosphamide); FR (fludarabine with rituximab);FCR (fludarabine, cyclophosphamide, and rituximab); CHOP(cyclophosphamide, doxorubicin, vincristine and prednisolone).

Allogeneic bone marrow (stem cell) transplantation—rarely used as afirst-line treatment for a B cell malignancy (e.g. CLL) due to its risk.There is increasing interest in the use of reduced intensity allogeneicstem cell transplantation, which offers the prospect of cure forselected patients with a suitable donor.

The ability of the antibodies of the present invention to bind CD84expressing B-CLL cells prompts their use in diagnostic applications.

Thus, according to an aspect of the invention there is provided a methodof diagnosing a B cell malignancy (e.g. B-CLL) in a subject in needthereof, the method comprising:

(a) contacting a biological sample of the subject with the antibody ofany one of claims 1-8 under conditions which allow the formation ofimmunocomplexes between CD84 isoform C (SEQ ID NO: 14) and saidantibody; and

(b) determining a level of said immunocomplexes in said biologicalsample, wherein an increase in level of said immunocomplexes beyond apredetermined threshold with respect to a level of said immunocomplexesin a biological sample from a healthy individual is indicative of the Bcell malignancy (e.g. B-CLL).

As used herein the term “diagnosis” or “diagnosing” refers toclassifying a pathology (e.g., cancer, e.g., B cell malignancy such asleukemia e.g., chronic lymphoid leukemia (CLL) e.g., B-CLL).

According to this aspect of the invention, the term “subject” or“subject in need thereof” refers to a mammalian e.g., human subjecthaving a routine check-up or screen for the pathology, as well as to asubject who is at risk of having the pathology such as due to familyhistory, environmental factors and/or a subject who exhibits suspiciousclinical signs of the pathology. Some clinical signs of a B cellmalignancy including B-CLL include but are not limited to predispositionto repeated infections such as pneumonia, herpes simplex labialis, andherpes zoster; enlarged lymph nodes; early satiety and/or abdominaldiscomfort which can be related to an enlarged spleen; mucocutaneousbleeding and/or petechiae which may be due to thrombocytopenia;tiredness and fatigue due to secondary to anemia; fevers, chills, andnight sweats and weight loss; autoimmune hemolytic anemia.

As used herein the phrase “CD84 isoform C” refers to the isoform of CD84which is assigned with Accession Numbers AF054815.1 NP_003865.1 (NM003874, Q9UIB8-3). SEQ ID NOs: 13, 14.

Examples of “biological samples” include but are not limited to wholeblood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, andvarious external secretions of the respiratory, intestinal andgenitourinary tracts, tears, saliva, milk as well as white blood cells,tissues, cell culture e.g., primary culture. According to a specificembodiment, the biological sample comprises B cells.

Malignant B cells (e.g. B-CLL cells) can be obtained from the blood, thebone marrow, the spleen, and/or the lymph nodes.

CD84 isoform C level can be determined at the protein level (level ofexpression and/or activity) or at the mRNA level (e.g., RT-PCR,real-time PCR etc.).

Following is a non-limiting list of examples of methods of determining alevel of CD84C using the antibody of the present invention.

Enzyme linked immunosorbent assay (ELISA): This method involves areaction between an enzyme and a substrate. A biological sample whichcomprises CD84C is put in a microwell dish. The CD84 specific antibodycoupled to an enzyme is applied and allowed to bind to the substrate.Presence of the antibody is then detected and quantitated by acolorimetric reaction employing the enzyme coupled to the antibody.Enzymes commonly employed in this method include horseradish peroxidaseand alkaline phosphatase. If well calibrated and within the linear rangeof response, the amount of substrate present in the sample isproportional to the amount of color produced. A substrate standard isgenerally employed to improve quantitative accuracy.

Western blot: This method involves separation of a substrate from otherprotein by means of an acrylamide gel followed by transfer of thesubstrate to a membrane (e.g., nylon or PVDF). Presence of the substrateis then detected by antibodies specific to the substrate (anti CD84antibody described herein is used), which are in turn detected byantibody binding reagents. Antibody binding reagents may be, forexample, protein A, or other antibodies. Antibody binding reagents maybe radiolabeled or enzyme linked as described hereinabove. Detection maybe by autoradiography, colorimetric reaction or chemiluminescence. Thismethod allows both quantitation of an amount of substrate anddetermination of its identity by a relative position on the membranewhich is indicative of a migration distance in the acrylamide gel duringelectrophoresis.

Radio-immunoassay (RIA): In one version, this method involvesprecipitation of the desired protein (i.e., the substrate) with a CD84specific antibody, as described herein, and radiolabeled antibodybinding protein (e.g., protein A labeled with I¹²⁵) immobilized on aprecipitable carrier such as agarose beads. The number of counts in theprecipitated pellet is proportional to the amount of substrate.

In an alternate version of the RIA, a labeled substrate and an unlabeledantibody binding protein are employed. A sample containing an unknownamount of substrate is added in varying amounts. The decrease inprecipitated counts from the labeled substrate is proportional to theamount of substrate in the added sample.

Fluorescence activated cell sorting (FACS): This method involvesdetection of a substrate in situ in cells by substrate specificantibodies i.e., CD84 antibody. The substrate specific antibodies arelinked to fluorophores. Detection is by means of a cell sorting machinewhich reads the wavelength of light emitted from each cell as it passesthrough a light beam. This method may employ two or more antibodiessimultaneously.

Immunohistochemical analysis: This method involves detection of asubstrate in situ in fixed cells by substrate specific antibodies, i.e.,anti CD84 antibody as described herein. The substrate specificantibodies may be enzyme linked or linked to fluorophores. Detection isby microscopy and subjective or automatic evaluation. If enzyme linkedantibodies are employed, a colorimetric reaction may be required. Itwill be appreciated that immunohistochemistry is often followed bycounterstaining of the cell nuclei using for example Hematoxyline orGiemsa stain.

As mentioned an increase in the level of the CD84C beyond apredetermined threshold with respect to the level of same in a similarsample from a healthy individual is indicative of the disease (e.g.,B-CLL).

As used herein, the phrase “biological sample from a healthy individual”refers to an unaffected control sample taken from a healthy subject(known not to have a B cell malignancy such as B-CLL) or from the samesubject prior to the onset of the B cell malignancy e.g. B-CLL (i.e.,healthy). Since biological characteristics depend on, amongst otherthings, species and age, it is preferable that the control saliva comefrom a subject of the same species, age. Alternatively, control data maybe taken from databases and literature. It will be appreciated that thecontrol sample may also be taken from the diseased subject at aparticular time-point, in order to analyze the progression (i.e.,monitoring) of the disease.

The term “increase” according to specific embodiment should bestatistically significant.

Once diagnosis is made, the subject may be informed of the disease i.e.,presence or absence of same and potential therapies for the B cellmalignancy e.g. B-CLL.

To improve assay sensitivity, the method may further comprisecorroborating the diagnosis using a diagnostic assay selected fromsurface marker expression distinctive of said CD84 isoform c, karyotypeanalysis and germline mutations.

Following is a non-limiting list of such assays/markers which can beused to corroborate the diagnosis of a B cell malignancy such as B-CLL.

Cell surface markers—B-CLL lymphocytes typically show B-cell surfaceantigens, as demonstrated by CD19, CD20, CD21, and CD23 monoclonalantibodies. In addition, they express CD5, which is more typically foundon T cells. Because normal CD5⁺ B cells are present in the mantle zone(MZ) of lymphoid follicles, B-CLL is most likely a malignancy of anMZ-based subpopulation of anergic self-reactive cells devoted to theproduction of polyreactive natural autoantibodies. B-CLL cells expressextremely low levels of surface membrane immunoglobulin, most oftenimmunoglobulin M (IgM) or IgM/IgD and IgD. Additionally, they alsoexpress extremely low levels of a single immunoglobulin light chain(kappa or lambda).

Genetic analysis—An abnormal karyotype is observed in the majority ofpatients with CLL. The most common abnormality is deletion of 13q, whichoccurs in more than 50% of patients. Individuals showing 13q14abnormalities have a relatively benign disease that usually manifests asstable or slowly progressive isolated lymphocytosis.

The presence of trisomy 12, which is observed in 15% of patients, isassociated with atypical morphology and progressive disease. Deletion inthe short arm of chromosome 17 has been associated with rapidprogression, short remission, and decreased overall survival in CLL.17p13 deletions are associated with loss of function of the tumorsuppressor gene p53. Deletions of bands 11q22-q23, observed in 19% ofpatients, are associated with extensive lymph node involvement,aggressive disease, and shorter survival.

More sensitive techniques have demonstrated abnormalities of chromosome12. Forty to 50% of patients demonstrate no chromosomal abnormalities onconventional cytogenetic studies. However, 80% of patients will haveabnormalities detectable by fluorescence in situ hybridization (FISH).Approximately 2-5% of patients with B-CLL exhibit a T-cell phenotype.

Investigations have also identified a number of high-risk geneticfeatures and markers that include germline immunoglobulin variable heavychain (IgV_(H)), IgV_(H) V3-21 gene usage, increased CD38 expression,increased Zap70 expression, elevated serum beta-2-microglobulin levels,increased serum thymidine kinase activity, short lymphocyte doublingtime (<6 mo), and increased serum levels of soluble CD23. These featureshave been associated with rapid progression, short remission, resistanceto treatment, and shortened overall survival in patients with B-CLL.

Germline mutations—Germline IgV_(H) has been shown to indicate a poorprognosis. Studies have shown that these patients also have earlierprogression of B-CLL after treatment with chemotherapy. The use ofcertain IgV_(H) genes, V3-21, have also been associated with poorprognosis regardless of IgV_(H) mutational status.

For any of the above clinical purposes (diagnostic or therapeutic), theanti CD84 antibody of the present invention can be bound (conjugated orattached) to a pharmaceutical agent.

Accordingly, the antibody can be attached to a pharmaceutical agent.

As used herein a pharmaceutical agent can be a drug (used in therapy) ora detectable moiety.

Various types of detectable or reporter moieties may be conjugated tothe proteins of the invention. These include, but not are limited to, aradioactive isotope (such as ^([125])iodine), a phosphorescent chemical,a chemiluminescent chemical, a fluorescent chemical (fluorophore), anenzyme, a fluorescent polypeptide, an affinity tag, and molecules(contrast agents) detectable by Positron Emission Tomography (PET) orMagnetic Resonance Imaging (MRI).

Examples of suitable fluorophores include, but are not limited to,phycoerythrin (PE), fluorescein isothiocyanate (FITC), Cy-chrome,rhodamine, green fluorescent protein (GFP), blue fluorescent protein(BFP), Texas red, PE-Cy5, and the like. For additional guidanceregarding fluorophore selection, methods of linking fluorophores tovarious types of molecules see Richard P. Haugland, “Molecular Probes:Handbook of Fluorescent Probes and Research Chemicals 1992-1994”, 5thed., Molecular Probes, Inc. (1994); U.S. Pat. No. 6,037,137 toOncoimmunin Inc.; Hermanson, “Bioconjugate Techniques”, Academic PressNew York, N.Y. (1995); Kay M. et al., 1995. Biochemistry 34:293; Stubbset al., 1996. Biochemistry 35:937; Gakamsky D. et al., “EvaluatingReceptor Stoichiometry by Fluorescence Resonance Energy Transfer,” in“Receptors: A Practical Approach,” 2nd ed., Stanford C. and Horton R.(eds.), Oxford University Press, UK. (2001); U.S. Pat. No. 6,350,466 toTargesome, Inc.]. Fluorescence detection methods which can be used todetect the antibody when conjugated to a fluorescent detectable moietyinclude, for example, fluorescence activated flow cytometry (FACS),immunofluorescence confocal microscopy, fluorescence in-situhybridization (FISH) and fluorescence resonance energy transfer (FRET).

Numerous types of enzymes may be attached to the antibody of theinvention [e.g., horseradish peroxidase (HPR), beta-galactosidase, andalkaline phosphatase (AP)] and detection of enzyme-conjugated antibodiescan be performed using ELISA (e.g., in solution), enzyme-linkedimmunohistochemical assay (e.g., in a fixed tissue), enzyme-linkedchemiluminescence assay (e.g., in an electrophoretically separatedprotein mixture) or other methods known in the art [see e.g., KhatkhatayM I. and Desai M., 1999. J Immunoassay 20:151-83; Wisdom G B., 1994.Methods Mol Biol. 32:433-40; Ishikawa E. et al., 1983. J Immunoassay4:209-327; Oellerich M., 1980. J Clin Chem Clin Biochem. 18:197-208;Schuurs A H. and van Weemen B K., 1980. J Immunoassay 1:229-49).

An affinity tag (or a member of a binding pair) can be an antigenidentifiable by a corresponding antibody [e.g., digoxigenin (DIG) whichis identified by an anti-DIG antibody) or a molecule having a highaffinity towards the tag [e.g., streptavidin and biotin]. The antibodyor the molecule which binds the affinity tag can be fluorescentlylabeled or conjugated to enzyme as described above.

Various methods, widely practiced in the art, may be employed to attacha streptavidin or biotin molecule to the antibody of the invention. Forexample, a biotin molecule may be attached to the antibody of theinvention via the recognition sequence of a biotin protein ligase (e.g.,BirA) as described in the Examples section which follows and inDenkberg, G. et al., 2000. Eur. J. Immunol. 30:3522-3532. Alternatively,a streptavidin molecule may be attached to an antibody fragment, such asa single chain Fv, essentially as described in Cloutier S M. et al.,2000. Molecular Immunology 37:1067-1077; Dubel S. et al., 1995. JImmunol Methods 178:201; Huston J S. et al., 1991. Methods in Enzymology203:46; Kipriyanov S M. et al., 1995. Hum Antibodies Hybridomas 6:93;Kipriyanov S M. et al., 1996. Protein Engineering 9:203; Pearce L A. etal., 1997. Biochem Molec Biol Intl 42:1179-1188).

Functional moieties, such as fluorophores, conjugated to streptavidinare commercially available from essentially all major suppliers ofimmunofluorescence flow cytometry reagents (for example, Pharmingen orBecton-Dickinson).

As used herein “drug” refers to a therapeutically active ingredient suchas a small molecule (e.g., chemotherapy), a toxin, a protein, a lipid, acarbohydrate or a combination of same.

Alternatively or additionally, the proteins can be attached (orconjugated) to non-proteinacious moieties which increase theirbioavailability and half-life in the circulation.

The phrase “non-proteinaceous moiety” as used herein refers to amolecule not including peptide bonded amino acids that is attached tothe above-described protein. Exemplary non-proteinaceous and preferablynon-toxic moieties which may be used according to the present teachingsinclude, but are not limited to, polyethylene glycol (PEG), Polyvinylpyrrolidone (PVP), poly(styrene comaleic anhydride) (SMA), and divinylether and maleic anhydride copolymer (DIVEMA).

Such a molecule is highly stable (resistant to in-vivo proteolyticactivity probably due to steric hindrance conferred by thenon-proteinaceous moiety) and may be produced using common solid phasesynthesis methods which are inexpensive and highly efficient, as furtherdescribed hereinbelow. However, it will be appreciated that recombinanttechniques may still be used, whereby the recombinant peptide product issubjected to in-vitro modification (e.g., PEGylation as furtherdescribed hereinbelow).

Thus, such non-proteinaceous non-toxic moieties may also be attached tothe above mentioned proteins to promote stability and possiblysolubility of the molecules.

Bioconjugation of such a non-proteinaceous moiety (such as PEGylation)can confer the proteins amino acid sequence with stability (e.g.,against protease activities) and/or solubility (e.g., within abiological fluid such as blood, digestive fluid) while preserving itsbiological activity and prolonging its half-life.

Bioconjugation is advantageous particularly in cases of therapeuticproteins which exhibit short half-life and rapid clearance from theblood. The increased half-lives of bioconjugated proteins in the plasmaresults from increased size of protein conjugates (which limits theirglomerular filtration) and decreased proteolysis due to polymer sterichindrance. Generally, the more polymer chains attached per peptide, thegreater the extension of half-life. However, measures are taken not toreduce the specific activity of the protein of the present invention(e.g., CD84 binding).

Bioconjugation of the protein with PEG (i.e., PEGylation) can beeffected using PEG derivatives such as N-hydroxysuccinimide (NHS) estersof PEG carboxylic acids, monomethoxyPEG₂-NHS, succinimidyl ester ofcarboxymethylated PEG (SCM-PEG), benzotriazole carbonate derivatives ofPEG, glycidyl ethers of PEG, PEG p-nitrophenyl carbonates (PEG-NPC, suchas methoxy PEG-NPC), PEG aldehydes, PEG-orthopyridyl-disulfide,carbonyldimidazol-activated PEGs, PEG-thiol, PEG-maleimide. Such PEGderivatives are commercially available at various molecular weights[See, e.g., Catalog, Polyethylene Glycol and Derivatives, 2000(Shearwater Polymers, Inc., Huntsville, Ala.)]. If desired, many of theabove derivatives are available in a monofunctional monomethoxyPEG(mPEG) form. In general, the PEG added to the CCL1 amino acid sequenceof the present invention should range from a molecular weight (MW) ofseveral hundred Daltons to about 100 kDa (e.g., between 3-30 kDa).Larger MW PEG may be used, but may result in some loss of yield ofPEGylated peptides. The purity of larger PEG molecules should be alsowatched, as it may be difficult to obtain larger MW PEG of purity ashigh as that obtainable for lower MW PEG. It is preferable to use PEG ofat least 85% purity, and more preferably of at least 90% purity, 95%purity, or higher. PEGylation of molecules is further discussed in,e.g., Hermanson, Bioconjugate Techniques, Academic Press San Diego,Calif. (1996), at Chapter 15 and in Zalipsky et al., “SuccinimidylCarbonates of Polyethylene Glycol,” in Dunn and Ottenbrite, eds.,Polymeric Drugs and Drug Delivery Systems, American Chemical Society,Washington, D.C. (1991).

Various conjugation chemistries of activated PEG such as PEG-maleimide,PEG-vinylsulfone (VS), PEG-acrylate (AC), PEG-orthopyridyl disulfide canbe employed. Methods of preparing activated PEG molecules are known inthe arts. For example, PEG-VS can be prepared under argon by reacting adichloromethane (DCM) solution of the PEG-OH with NaH and then withdi-vinylsulfone (molar ratios: OH 1: NaH 5: divinyl sulfone 50, at 0.2gram PEG/mL DCM). PEG-AC is made under argon by reacting a DCM solutionof the PEG-OH with acryloyl chloride and triethylamine (molar ratios: OH1: acryloyl chloride 1.5: triethylamine 2, at 0.2 gram PEG/mL DCM). Suchchemical groups can be attached to linearized, 2-arm, 4-arm, or 8-armPEG molecules. It will be appreciated that the antibodies of theinvention may be produced using recombinant DNA technology (where apolynucleotide encoding the antibody of the invention is introduced intoan appropriate host cell where the antibody is synthesized. Exemplarysequences are provided in SEQ ID NOs: 26 and 27) or by chemicalsynthesis such as by solid phase techniques.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

When reference is made to particular sequence listings, such referenceis to be understood to also encompass sequences that substantiallycorrespond to its complementary sequence as including minor sequencevariations, resulting from, e.g., sequencing errors, cloning errors, orother alterations resulting in base substitution, base deletion or baseaddition, provided that the frequency of such variations is less than 1in 50 nucleotides, alternatively, less than 1 in 100 nucleotides,alternatively, less than 1 in 200 nucleotides, alternatively, less than1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides,alternatively, less than 1 in 5,000 nucleotides, alternatively, lessthan 1 in 10,000 nucleotides.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate some embodiments of the invention in anon limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Materials and Experimental Procedures

Transfection and protein expression of secreted CD84-ECD: For expressionand secretion of CD84-ECD protein (SEQ ID NO: 15), HEK 293T cells wereplated in 175 cm³ cell culture flasks to a final confluence of 75% 24 hbefore transfection. For transfection, 30 mL complete DMEM medium and 3mL of DNA precipitate-containing solution were used. Cultures wereincubated an additional 24 hours after transfection, and then the mediumwas changed to serum-free DMEM for 2 days. The conditioned medium wascollected; centrifuged for 1 h at high speed to remove cells and debris,filtrated with 0.2 μm filter unit and protein purification fromconditioned medium was done using FLPC (Nickel affinity chromatography).After binding of the protein to the Ni2+ column, it was washed andeluted with high concentrations of imidazole, which competes with theHis6-tag and displaces the protein. After purification, concentratedsamples from each purification step and from the different peaks wereanalyzed by SDS-PAGE followed by western blotting and Coomassie stainingof the gel.

Size-exclusion chromatography (gel filtration): The pooled, concentratedfractions of the Ni2+ affinity chromatography were loaded onto a 16/60Superdex 200 column (preparative grade) by injection into the FPLCsystem. The proteins were eluted at a flow rate of 5 mL/min with PBS.Fractions were collected according to the UV absorption, and changes inpH of the elutes. After pooling and concentrating the different peakfractions, samples were analyzed by SDS-PAGE, followed by westernblotting and Coomassie staining.

Protein identification by ESI-MS/MS: In order to verify the identity ofthe CD84-ECD protein, the presumptive band of the Coomassie-stained gelwas excised and analyzed by proteolytic digestion followed byelectrospray ionization mass spectrometry. The procedure was carried outby the Biological Service Unit at the Weizmann Institute.

Hybridoma protocol: CD84-ECD protein was purified (as described before)from conditioned medium derived from 293 cells transfected with theCD84-ECD construct. Mice were immunized with purified CD84-ECD over aperiod of 3 months. Following positive ELISA test bleeding forantibodies against CD84-ECD (the plates were coated with purifiedECD-CD84), spleens were removed; lymphocytes were isolated (with HBSS)and fused with NSO cells (ratio of 1:5) together with polyethilenglycol(PEG). Hybridomas were selected with HAT (Hypoxantine AminopterineTymidine) medium and their supernatant was analyzed for recognition ofCD84-ECD using ELISA assay [Ho M K, Springer T A. Methods Enzymol 1984;108: 313-324].

ELISA for hybridoma subclass detection: Hybridomas were shown to be ofthe IgM, k class by commercial ELISA kit. Briefly, cell-free culturesupernatants were harvested from hybridoma, and IgM, IgG3, IgG1, αb, αa,Δ,λ,κ levels were determined using an ELISA methods according tomanufacturer's instructions (BD Bioscience).

Hybridoma activation/blocking: Hybridoma B1/B4/F8 was added to CLL cellsgrowing in RPMI medium containing 10% (v/v) FCS incubated at 37° C. for18 h (for RNA extraction) and 24 h (for protein extraction), with orwithout MIF stimulation.

CLL Patient population: B lymphocytes were taken from the peripheralblood of both healthy subjects (normal/control) and CLL patients whosatisfied diagnostic and immunophenotype criteria for CLL, at variousstages of the disease. Cells were provided by the Hematology Instituteof Kaplan Medical Center and the Sourasky Medical Center, in accordancewith the IRB of the hospital, as described previously [Haran M et al.,Leukemia. 2004; 18:1948-1950]. The diagnosis of CLL was based onstandard criteria and patients were staged according to the Rai stagingsystem [Hallek M et al., Blood. 2008; 111:5446-5456].

Cell purification: B-lymphocytes were purified using a RosettSepantibody cocktail (StemCell Vancouver, BC, Canada) as previouslydescribed [Lantner F et al., Proc. Natl. Acad. Sci. U.S.A. 2007;104:13408-13413]. Briefly, this method is based on negative selection ofB cells. 50 μl of cocktail containing divalent antibodies targeted atnon-B-cell antigens (CD3, CD2, CD16, CD56, and CD14) was added to eachmilliliter of whole blood. After an incubation of 20 min, an equalamount of PBS was added to the mixture and the cells were put on aFicoll gradient, centrifuged and isolated from the ficoll by PBS washes.Cells were used fresh or viably frozen in fetal calf serum (FCS) plus10% dimethyl sulfoxide (DMSO) for storage in liquid nitrogen. Frozencells were cultured overnight in 5% CO₂ in RPMI with 10% FCS andantibiotics.

M210B4 cells: M2-10B4 cells were purchased from ATCC (ATCC® CRL-1972™).This cell line is a clone derived from bone marrow stromal cells from a(C57BL/6J×C3H/HeJ)F1 mouse. Cells were grown in RPMI-1640 Medium,supplemented with 10% FCS and penicillin-streptomycin-glutamine andcultured in 37° C. in 5 % CO₂.

Cells of solid tumors: MDA435 (Human breast cancer), PC-3 (Humanprostate cancer), Hela (Human cervical cancer), A375 (Human malignantmelanoma; CRL 1619), A549 (Human lung adenocarcinoma), PAC1 (HumanPancreas cancer; 39532) and NCL-N87 (Human gastric cancer) werepurchased from ATCC. Cells were grown in DMEM Medium, supplemented with10% FCS and penicillin-streptomycin-glutamine and cultured in 37° C. in5% CO₂.

Daudi cells: The Burkitt's lymphoma cell line, Daudi, was purchased fromATCC (ATCC® CCL-213™). 1×10⁶ Daudi cells were cultured in RPMI mediumcontaining 10% FCS with different concentrations of the anti-CD84antibodies B1 or B4 or with a control antibody. Following 48 hours, cellsurvival was examined using Annexing staining (as described below).

Antibodies: For FACS staining anti CD84-PE (CD84.1.21 Bio Legend). Forblocking: LEAF™ Purified anti-human CD84 Antibody (CD84.1.21 BioLegend).

Flow cytometry: Staining of CLL cells was performed as previouslydescribed [Binsky I et al., Proc. Natl. Acad. Sci. U.S.A. 2007;104:13408-13413]. The following antibodies were used: CD-19(MiltenyiBiotec, Auburn, Calif.), anti-CD74 (Santa Cruz), IgG1κ isotypecontrol (MOPC-21, BD Biosciences), CD49d (9F10, BD Biosciences),anti-CD84 (Ab-2, 152-1D5 Labvision), followed by PE anti-mouse secondaryantibody (Jackson ImmunoResearch Laboratories) or anti CD84-PE(CD84.1.21 Bio Legend), PE anti-human CCR1/CCR5 Antibody (BioLegend), PEanti-mouse IgM Antibody, RMM-1 (BioLegend), APC anti-mouse/humanCD45R/B220 Antibody, RA3-6B2 (BioLegend), Anti-Mouse CD5 eFluor®, 45053-7.3 (eBioscience). Staining was analyzed by FACSCanto (BDBiosciences).

Intracellular Staining for Bcl-2: Cells were permeabilized and fixedusing BioLegend's FOXP3 Fix/Perm solution for 20 minutes in roomtemperature, than washed twice with cell staining buffer and again withBioLegend's FOXP3 Perm buffer. Next, cells were incubated withBioLegend's FOXP3 Perm buffer for 15 minutes in the dark and thenstained for 30 min on ice with anti-PE anti-Bcl-2 Antibody (BCL/10C-4,Biolegend) or anti-isotype control. Cell staining was assessed inFACSCanto flow cytometer (BD Biosciences).

Cell death detection: Annexin and PI/7AAD staining: Purified CLL cellswere cultured in 24-well plates at 1×10⁷ cells/well in RPMI mediumsupplemented with 10% FCS, with or without 1.5-2 μg of anti-CD84(ab-3202 Abcam/152-1D5 Thermo) followed by 0.5 μg of anti Fab (Jackson)or an IgG isotype control antibody (MOPC-21 BioLegend) for 24 and 48 h.Cells were centrifuged, washed, and stained with Annexin (BDBiosciences), and propidium iodide (PI; 25 μg/ml) (Sigma) or 7AAD (BDBiosciences) was added for 15 min at room temp in the dark. The extentof Annexin+PI/7AAD staining was analyzed by FACSCanto (BD Biosciences).

Magic Red Apoptosis Detection Kit: 10⁷ CLL cells per ml were incubatedat 37° C. for 1 h with Magic Red (Magic Red Caspase Detection Kit forcaspase 3 and 7 MR-(DEVD)2 Immunochemistry Technology) according to themanufacturer's instructions. Then, Magic Red staining was measured byFACSCalibur (BD Biosciences) analysis.

Enzyme-linked immunosorbent assay (ELISA): CLL cells were cultured withanti CD84 agonist or blocking antibody or B4 hybridoma, as previouslydescribed for 48-72 h. Co-cultures of CLL cells and NLC/M210B4 cellswere blocked for CD84 using an antagonist antibody or blocking hybridomaB4 as described before.

Cell-free cultures supernatants were then harvested and levels of hCCL3and mIL6 were determined using an ELISA method according tomanufacturer's instructions (Human CCL3/mouse IL6 ELISA DuoSet R&Dsystems).

Blocking CD84 in co-cultures of CLL and M210B4: 1×10⁵ stromal cells wereplated per well in 24-well plate. After 18 hr, 1.6×10⁶ CLL cells wereadded into each well, in the presence or absence of anti-CD84 blockingantibody (5 μg/μl) or B4 hybridoma (2.5-5 μg/μl) or isotype control andco-cultured for another 48 hr for viability assay of CLL cells or 72 hfor ELISA of CCL3.

CD84 blocking: was performed as previously described [Binsky-Ehrenreichet al. E. Pub. Feb. 25, 2013 Oncogene]. Briefly, 1×10⁷ CLL cells wereincubated in RPMI medium containing 0.1% (v/v) FCS at 37° C. for 3 h.Next, cells were resuspended in medium containing 2.5 μg of anti CD84(CD84.1.21 BioLegend) or by B4 hybridoma 2.5-5 μg (see material andmethods) and incubated at 37° C. for 18 h (for RNA extraction) and 24-72h (for protein extraction, intracellular staining and ELISA).

Real-time reverse transcription-PCR analysis: Levels of mRNA of humanCD84, Rp2, Bcl-2, CCL3, IL6, and VCAM were analyzed by quantitativereal-time RT-PCR using a Light-Cycler instrument (Roche Diagnostics,Mannheim, Germany). The reaction volume (10 μl) contained 3 mM MgCl2,LightCycler HotStart DNA SYBR Green I mix (Roche Diagnostics), specificprimer pairs, and 2.5 μl of cDNA. Conditions for PCR were as follows: 10minutes at 95° C. followed by 40-60 cycles of 15 seconds at 95° C., 15seconds at 60° C., and 15 seconds at 72° C. PCR was performed induplicates as previously described [Luger D et al., J. Clin. Immunol.2004; 24:579-590].

Primer sequences were as follows:

-   hBcl-2: 5′GGATCAGGGAGTTGGAAG3′ GCACTGCCAAACGGAG (SEQ ID NOS: 16-17,    respectively).-   hCCL3: 5′CGAGCAGCCAGTGCTCCAAGC 3′GGCTGTTTGGCAACAACCAGT CCA (SEQ ID    NOs: 18-19, respectively).-   Mouse Bcl-2: 5′ GCTACCGTCGTGACTT 3′ GCCGGTTCAGGTACTC (SEQ ID NOs:    20-21, respectively).-   IL6 mouse: 5′ CCACTTCACAAGTCGGAGGCT TA 3′GCAAGTGCATCATCG TTGTTCATAC    (SEQ ID NOs: 22-23, respectively).-   VCAM1 mouse: 5′ TACCAGCTCCCAAAATCCTG 3′TCTGCTAATTCCAGCCTC GT (SEQ ID    NOs: 24-25, respectively).-   RP-2 levels were used to normalize samples for calculation of the    relative expression levels of other genes.-   CD84: 5′TTGTTCCGTTTGTTCAAGAG 3′CGGAATAAACTGTGTTCACTG^(h) (SEQ ID    NOs: 28-29, respectively).

Downregulation of CD84 by siRNA: ON-TARGETplus SMARTpool, Human CD84(NM_003874), Dharmacon, was used for downregulation of CD84.

Mice: All animal procedures were approved by the Animal ResearchCommittee at the Weizmann Institute.

TCL1 mice as a model for chronic lymphocytic leukemia: The percent ofmalignant population in various ages of TCL-1 mice were determined.Splenic cells were obtained from 18, 11, and 6 months old TCL-1 mice andwere analyzed for their B220+CD5+ population by FACSCanto (BDBioscience).

Splenic B cells were then incubated with anti-CD84 (B1 or B4) or anisotype matched control antibody in Iscove's medium containing 2% FCSfor 48 h. Cell survival was analyzed by Annexin-7AAD staining (asdescribed above).

Xenograft model for CLL: CLL cells were labeled with 5 μMcarboxyfluorescein diacetate succinimidyl ester (CFSE; Anaspec) for 5min at 37° C. CFSE had no effect on cell survival throughout theexperiment. 1×10⁷ cells then were i.v. injected into control C57BL/6 andCD84 KO mice. Homing of CLL cells to the spleen, BM and PB wasdetermined 1 h and 4 h later by FACS analysis of isolated cells fromtissues.

Normal B cells: Splenic B cells were obtained from 6, 8 and 11 monthsold C57BL/6 mice. Cells were isolated and stimulated with 5 μg/mlanti-CD84 B1 or B4 antibodies, and compared to the effect of an isotypematched control antibody.

Eμ-TCL1 model for CLL: Eμ-TCL1 transgenic mice were used to follow CLLprogression by monitoring CD5⁺ B lymphocytes expansion as previouslydescribed [Bichi R et al., Proc Natl Acad Sci USA. 2002. 99: 6955-6960].Chimeric mice were generated by transplanting WT and CD84 KO mice with5×10⁶ BM cells from Eμ-TCL1 mice. After 4, 6, 8 and 13 months theperitoneal cavity, PB, Spleen and BM of the recipient mice weresacrificed and cells were extracted, stained for CD5 (450 53-7.3(eBioscience), IgM (RMM-1 BioLegend), and B220 (RA3-6B2 (BioLegend) andanalyzed by FACSCanto (BD Bioscience).

Example 2 The B4 Antibody Induces Apoptosis in CLL Cells

The present inventors have determined whether the B4 antibodiesgenerated according to the present teachings are capable of inducingapoptosis in primary human CLL cells. As demonstrated in FIGS. 1A-D, thehybridoma-derived B4 antibody, induced cell death in CLL cells culturedalone (FIGS. 1A-B). As previously shown, different types of stromalcells, such as primary human Nurse like cells (NLC) and marrow stromalcells (MSC) (cell line M210B4, CRL-1972™) protect CLL cells inco-culture from apoptosis, and are an integral part of the CLLmicroenvironment (Burger J A et al., Blood. 2009. 114:3367-3375, KurtovaA V et al., Blood. 2009. 114:4441-4450). Interestingly, the B4hybridoma-derived antibody overcame the protective effect of the stromaand induced CLL death when co-cultured with M210B4 cells (FIGS. 1C-D).Therefore B4 hybridoma, can be used as a blocking antibody. Measuring ofapoptosis includes all annexin positive cells (single and double)annexin positive total population increases from 46.18% to 55.75%.

Example 3 CD84 Regulates an Anti-Apoptotic Effect on Bone Marrow StromalCells-B4

It has been previously suggested that CLL cells are able to activelymanipulate their microenvironment [Binder M et al, PLoS One. 2012.5(12):e15992, Neil E Kay et al, Leuk Res. 2007 July; 31(7): 899-906].Specifically, it has recently been shown that CLL cells induceanti-apoptotic effects on their stromal counterparts in culture. TheCLL-stoma cell interaction induces the expression and secretion ofspecific cytokines (i.e IL-6, IL-8) from the stroma and induces theexpression of specific adhesion molecules like ICAM-1, while theexpression of other adhesion molecules remains unchanged (i.e VCAM-1)[Plander M et al, Annals of Hematology. 2011. 90(12):1381-90].

To determine whether the CLL-stromal cells interaction mediated by CD84induces a similar cascade in the stroma, CLL cells were cultured withM210B4 stomal cells in the presence or absence of a CD84 blockinghybridoma (B4) as described above (FIGS. 1C-D), and expression of theanti-apoptotic gene Bcl-2 (FIG. 2A), the cytokine IL-6 (FIG. 2B), andVCAM-1 (FIG. 2C) in M210B4 cells were monitored. Incubation with CLLcells elevated Bcl-2 and IL-6 mRNA levels in M210B4 cells, while nochange in VCAM-1 message were observed.

Interestingly, CD84 blockage reduced Bcl-2 (FIG. 2A) and IL-6 (FIG. 2B)message levels, while VCAM-1 levels were not affected by this blockage(FIG. 2C).

Example 4 CD84 Regulates CCL3 Expression in Co Cultures-B4

It was previously shown that CLL cells are able to actively recruitcells and create a supportive microenvironment via the secretion of CCL3[Zucchetto A et al., Cancer Res. 2009. 69(9):4001-4009. Burger J A etal., 2009. 113:3050-3058]. Stimulation of CD84 expressed on CLL cellsinduces CCL3 expression and secretion (our non-published results). Todirectly demonstrate the regulatory role of CD84 in the CCL3 mediatedsecretion in CLL-stromal cells co-culture, CCL3 secreted levels wereanalyzed in the supernatants derived from CLL-stromal cells co-cultures,which were incubated in the presence or absence of a CD84 blockinghybridoma (B4) or an isotype control antibody. As demonstrated in FIG.3, blocking of CD84 in CLL-M210B4 co-culture, downregulated the levelsof human CCL3, derived from the CLL cells, secreted to the conditionedmedium as detected by ELISA (FIG. 3).

Similarly, CCL3 secretion from CLL cells co-cultured with NLC cells inthe presence or absence of anti-CD84 blocking antibody was analyzed.Blocking CD84 significantly downregulated secretion of CCL3 from CLLcells to the conditioned medium (data not shown). However, as NLC are ofhuman origin, it was further verified that CD84 specifically regulatessecretion of CCL3 from CLL cells in these co-cultures. Therefore CCL3mRNA levels were analyzed in CLL cells cultured with NLC cells anddemonstrated reduced expression of CCL3 message in CLL cells (data notshown).

Thus, CLL-microenvironment interaction is mediated by CD84, whichpromotes secretion of CCL3 from CLL cells.

Example 5 Comparison Between F8, B1 and B4 Antibodies

As shown in FIGS. 4A-H and in Table 1 below, antibodies of someembodiments of the invention (B1 and B4) were able to superiorly killpurified CLL cells, as demonstrated by higher percentage of apoptoticcells (FIGS. 4A-D), and Ramos (RA-1) Burkitt's lymphoma cells (ATCCCRL-1596™, FIGS. 4E-H) as compared to a blocking antibody described inthe art (F8, PCT publication no. WO2010/035259).

TABLE 1 Comparison of apoptosis of CLL cells by F8, B1 and B4 antibodiesPurified CCL Ramos Cells (% apoptosis) (% apoptosis) F8 34 16 B1 73 45B4 75 39

Example 6 CD84KO Mice Show Decreased Number of Human CLL Cells in TheirBM

In order to study the survival and the distribution of human CLL cellsin the different compartments of transplanted mice, human primary CLLcells were fluorescently labeled with CFSE. The cells were then injectedto CD84KO and WT mice. Following 4 hours, the number of cells residingin the different compartments was compared, and the ratio between thenumbers of cells in the BM and the spleen of each mouse was calculated.As shown in FIGS. 5A-B, the ratio of CLL cells detected in the BM of thecontrol mice was significantly reduced in CD84KO mice (FIG. 5A). Inorder to determine whether this phenomenon is a result of impairedarrival of the CLL cells to the BM compartment in CD84KO mice, or due totheir inability to retain or survive in this compartment, CLL cells wereinjected to CD84KO or WT mice and the mobilization of cells to the BMwas followed 1 hour after injection. As shown in FIG. 5B, homing of CLLcells to the BM after 1 hour was similar in WT and CD84 KO mice (FIG.5B). This result might suggest that CLL cells home normally to the BMwhich lacks CD84 expression in the microenvironment. The reduced numberof cells detected in the BM at a later time point is possibly due to theinability of the cells to retain or survive in the BM microenvironmentin the absence of the CD84 signal.

Example 7 Evaluation of Species Cross Reactivity

Eμ-TCL1 a transgenic mouse model of CLL disease was used. In Eμ-TCL1transgenic mice, there is an accumulation of B220 low IgM+CD5+ Blymphocytes in the peritoneal cavity, peripheral blood, bone marrow andlymphoid organs of the mice. The IgM+CD5+ B lymphocytes express CD84 ontheir surface (FIG. 6A).

It was then determined whether blocking CD84 activity with the B1 and B4antibodies induced cell death. First, the percent of the malignantpopulation in various ages of TCL-1 mice were determined. Splenic cellsderived from 18, 11, and 6 months old TCL-1 mice were analyzed for theirB220+CD5+ population by FACS. While most splenic cell from the 18 and 11months old TCL-1 mice were the malignant cells (B220+CD5+), theB220+CD5+ population could hardly be detected in spleens derived from 6months old TCL-1 mice (FIGS. 6B-D). Next, the effect of the antibodieson cell survival was followed. The malignant splenic B cells wereincubated with anti-CD84 (B1 or B4) or an isotype matched controlantibody in Iscove's medium containing 2% FCS. 48 h later cell survivalwas analyzed by Annexin-7AAD staining. As can be seen in FIGS. 6E-M,blocking CD84 with B1 and B4 antibodies induced apoptosis in themalignant B220+CD5+ population. Surprisingly, survival of peripheralnon-malignant B220+CD5− B cell population was not affected by thistreatment (as described in FIGS. 9A-J). These results suggest that theanti human CD-84 antibodies, B1 and B4, can block murine CD84 onmalignant mouse cells and reduce their survival.

Example 8 Analysis of CD84 Expression on Tumors

Various cell lines express CD84 as is illustrated in the websitewww.broadinstitute.org/ccle/. Most of the cell lines belong to thehematopoietic origin, however, a few solid tumors were selected toanalyze CD84 expression. mRNA was extracted from different solid tumorcell line: MDA435 (Human breast cancer), PC-3 (Human prostate cancer),Hela (Human cervical cancer), A375 (Human malignant melanoma), A549(Human lung adenocarcinoma), PAC1 (Human Pancreas cancer) and NCL-N87(Human gastric cancer). CD84 message was analyzed by RT-PCR. As can beseen in FIG. 7A, CD84 mRNA was not detected in any of these cells.

Analysis of the CD84-induced cascade in CD84 expressing hematopoietictumor cells was carried out in the Burkitt's lymphoma cell line, Daudi.As can be seen in FIG. 7B, Daudi cells express CD84 on their cellsurface.

Next, the effect of the anti-CD84 B1 and B4 antibodies on apoptosisinduction in Daudi cells was determined. Daudi cells were cultured withdifferent concentrations of the anti-CD84 antibodies B1 or B4 or with acontrol antibody. Following 48 hours, cell survival was examined usingAnnexing staining. Blocking CD84 induced death of the Daudi cells (arepresentative concentration is shown in FIG. 7C).

Example 9 Analysis of CD84 Expression and Function in Normal Tissues

Various normal tissues express CD84 as is illustrated in the websitewww.gtexportal.org/home/. As shown therein, CD84 is expressed mainly onEBV transformed cells and in the spleen.

The role of CD84 as a survival receptor in normal tissues was analyzed.CD84 expression levels in healthy and CLL cells from patients at variousdisease stages were compared. Purified B cells from healthy subjects aswell as early- and advanced-stage CLL cells were analyzed for thepresence of CD84 mRNA (targeting a segment common to all isoforms). Asshown in FIG. 8A-B, low levels of CD84 mRNA were detected in normal Bcells, while elevated levels of CD84 mRNA were observed in all the CLLpatients, regardless of disease stage.

In addition, CD84 cell surface levels were significantly higher in allCLL cells when compared to total (FIGS. 8C-E) or CD5+ normal B cells(FIG. 8F). Thus, normal B cells express CD84 at lower levels.

Next, the effect of anti-CD84 B1 and B4 antibodies on survival of normalB cells was evaluated. Splenic B cells derived from 6, 8 and 11 monthsold C57BL/6 mice were isolated and stimulated with 5 μg/ml anti-CD84 B1or B4 antibodies, and compared to the effect of an isotype matchedcontrol antibody. As can be seen in FIGS. 9A-J, both B1 and B4 had noeffect on the viability of peripheral normal B cells. These resultssuggest that while B1 and B4 antibodies can block murine CD84 activityon malignant mouse cells and reduce cell survival (FIG. 6C), they do notinduce apoptosis in normal B cells.

Example 10 Analysis of CD84 Expression in the Tumor Microenvironment

CD84 expression on marrow stromal cells (MSCs) and nurse like cells(NLC) present in the tumor microenvironment was followed. For MSCs,primary bone marrow MSCs derived from CLL patients were used, thesecells were isolated and cultured as previously described [Kay et al.,Leukemia Research 31, 899-906 (2007)] and murine MSC lines (M210B4) F1mouse were also used. These cell lines were shown to protect CLL cellsfrom spontaneous and drug-induced apoptosis [Kurtova et al., Blood 114,4441-4450 (2009)]. NLC were isolated and cultured as describedpreviously [Burger et al., Blood 96, 2655-2663 (2000)].

As previously shown CD84 mRNA was detected in CLL cells (FIG. 10A). Inaddition, CD84 mRNA was detected in M210B4 cells as well as in NLC (FIG.10A). Next, CD84 expression was analyzed on the surface of these cells.As shown in FIG. 10B, in addition to its expression on CLL cells, CD84was detected on the surface of NLC as well as M210B4 stromal cells. Thissuggests that CD84 might be involved in cell-cell interaction betweenCLL cells and their microenvironment.

Example 11 Analysis of the Role of CD84 in the Regulation ofMicroenvironment-Induced Protection from CLL Spontaneous andDrug-Induced Apoptosis

To determine whether CD84 plays a role in the interaction of CLL cellswith their microenvironment which results in cell survival, CLL cellswere co-cultured with stroma cells (as described above) in the presenceor absence of anti-CD84 blocking antibodies. M210B4 (FIGS. 11A-G)induced an anti-apoptotic effect on CLL cells, as measured byAnnexin-PI/7AAD staining (described in detail in Example 1 above).Blocking of CD84, by the hybridoma B4 (FIGS. 11A-C), partially overcamethe anti-apoptotic effect induced by the stroma, resulting in inductionof CLL cell death.

The present inventors next wanted to determine whether the apoptosisinduced by blocking CD84 was a direct effect on CLL cells, or if theantibodies could prevent the interaction of the CLL cells with thestroma, thereby inducing their death. Thus, CD84 expression wasdownregulated in M210B4 cells by siRNA. CLL cells were then added to theculture and their survival was monitored. As demonstrated in FIG. 11D-G,deletion of CD84 specifically reduced the stroma-mediated survival ofCLL cells. Thus, CD84 expressed on the stroma mediates survival of CLLcells via cell to cell contact. To further demonstrate the role of CD84expressed on the microenvironment on survival of CLL cells, CD84expressed on M210B4 was blocked by the B1 and B4 antibodies. After 1hour, the antibodies were washed and CLL cells were then added to theco-culture. 48 hours later cell survival was measured by Annexin-7AADstaining. Blocking of stromal CD84 significantly induced apoptosis ofCLL cells (FIGS. 12A-D), suggesting that the survival induced by thestroma is mediated by CD84 stromal-CLL interaction.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

1. An isolated antibody comprising an antigen recognition domain whichspecifically binds CD84 and (i) down regulates the anti-apoptoticactivity of stromal cells on chronic lymphocytic leukemia (CLL) cells;and/or (ii) induces mobilization of CLL cells from the bone marrow. 2.An isolated antibody comprising an antigen recognition domaincomprising: complementarity determining regions as set forth in SEQ IDNOs: 1, 2, 3, 4, 5 and 6 (B4); or complementary determining regions asset forth in SEQ ID NOs: 7, 8, 9, 10, 11 and 12 (B1), wherein theantibody specifically binds CD84.
 3. (canceled)
 4. The isolated antibodyof claim 2, down regulating the anti-apoptotic activity of stromal cellson chronic lymphocytic leukemia (CLL) cells.
 5. The isolated antibody ofclaim 2, inhibiting the secretion of CCL3 from CLL cells.
 6. Theisolated antibody of claim 2, inhibiting the expression of BCL-2 in CLL.7. The isolated antibody of claim 2, inhibiting the expression of stromaBCL-2.
 8. The isolated antibody of claim 2, reducing the expression ofIL-6 and IL-8 in stromal cells.
 9. The isolated antibody of claim 2,inducing mobilization of CLL cells from the bone marrow.
 10. Theisolated antibody of claim 2, being an IgG.
 11. The isolated antibody ofclaim 2, wherein the antibody is a humanized antibody or a chimericantibody.
 12. The isolated antibody claim 2, wherein the antibody is abispecific antibody.
 13. A pharmaceutical composition comprising theisolated antibody of claim 2 and a pharmaceutically acceptable carrieror diluent.
 14. A method of inducing apoptosis in B cells of a subjecthaving a B cell malignancy, the method comprising administering to thesubject a therapeutically effective amount of the antibody of claim 2,thereby inducing apoptosis in B cells of the subject.
 15. A method oftreating a B cell malignancy in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of the antibody of claim 2, thereby treating the B cellmalignancy.
 16. (canceled)
 17. The method of claim 14, wherein said Bcell malignancy is selected from the group consisting of a lymphoma, aleukemia and a myeloma.
 18. (canceled)
 19. A method of diagnosing B-CLLin a subject in need thereof, the method comprising: (a) contacting abiological sample of the subject with the antibody of claim 2 underconditions which allow the formation of immunocomplexes between CD84isoform C (SEQ ID NO: 14) and said antibody; and (b) determining a levelof said immunocomplexes in said biological sample, wherein an increasein level of said immunocomplexes beyond a predetermined threshold withrespect to a level of said immunocomplexes in a biological sample from ahealthy individual is indicative of the B-CLL.
 20. The method of claim19, wherein said determining is effected at the protein level.
 21. Themethod of claim 19, further comprising corroborating the diagnosis usinga diagnostic assay selected from surface marker expression distinctiveof said CD84 isoform c, karyotype analysis and germline mutations. 22.An isolated polynucleotide comprising a nucleic acid sequence encodingthe antibody of claim
 2. 23. The isolated polynucleotide of claim 22 asset forth in SEQ ID NO: 26 or
 27. 24. A method of identifying a putativedrug against a B cell malignancy, the method comprising: (a) treating amodel animal having B cell malignancy with a test agent; and (b)detecting B cell malignancy cells in a peripheral tissue versus in abone marrow of said model animal treated as in (a), wherein an increasein said ratio as compared to said ratio prior to said treatment isindicative that said test agent is a putative drug against B cellmalignancy.
 25. The method of claim 24, wherein said test agent is ananti CD84 antibody.
 26. The method of claim 24, wherein said B cellmalignancy is B-CLL.
 27. The method of claim 24, wherein said modelanimal is selected from the group consisting of a xenograft modelinduced by transplanting human CLL in mice and a Eu-TCL1 murine model.28. A pharmaceutical composition comprising the isolated antibody ofclaim 1 and a pharmaceutically acceptable carrier or diluent.